2.1. USE OF HEMOGLOBIN AS A BLOOD SUBSTITUTE
Transfusion of a patient with donated blood has a number of disadvantages. Firstly, there may be a shortage of a patient's blood type. Secondly, there is a danger that the donated blood may be contaminated with infectious agents such as hepatitis viruses, cytomegalovirus, Epstein-Barr virus, serum parvoviruses, syphilis, malaria, filariasis, trypanosomiasis, babsiosis, pathogenic bacteria, and HIV (Bove, 1986, Progr. Hematol. 14:123-145). Thirdly, donated blood has a limited shelf life.
An alternative to transfusion involves the use of a blood substitute. A blood substitute is an oxygen carrying solution that also provides the oncotic pressure necessary to maintain blood volume. Two types of substitutes have recently been studied, fluorocarbon emulsions and hemoglobin solutions.
Hemoglobin as it exists within the red blood cell is composed of two alpha-like globin chains and two beta-like globin chains, each with a heme residue. One alpha-like globin chain and one beta-like globin chain combine to form a dimer which is very stable. Alpha-like and beta-like globin genes are each a family of related globin genes which are expressed at different stages of development and regulated by oxygen tension, pH, and the development from embryo to fetus to newborn. Two dimers then line up in antiparallel fashion to form tetramers. The binding of dimers to form the tetramers is not as strong as in the case of monomers binding to associate into dimers. The tetramers, therefore, have a tendency to fall apart to form dimers and there is always an equilibrium between tetramers, dimers, and monomers. At high concentrations of globin, the predominant form is the tetramer; with dilution, the dimer becomes the predominant form. This equilibrium is also affected by solvent, salts, pH and other factors as the forces binding the monomers together are primarily electrostatic.
The alpha-like globin genes are clustered together on chromosome 16 and include genes encoding the embryonic zeta globin chain and the adult alpha globin chain, present in both the fetus and newborn. The beta-like globin genes reside on chromosome 11 and include genes encoding the embryonic epsilon-globin chain, the fetal gamma-globin chain, and the adult delta-globin and adult beta-globin chains. Two types of gamma-globin chains have been identified, .sup.G gamma and .sup.A gamma, which differ by the presence of a single glycine or alanine residue, respectively, at amino acid 135 (Schroeder et al., 1968, Proc. Natl. Acad. Sci. U.S.A. 60: 537-544). The gamma-globin chain has been found to contain a polymorphic site at position 75, which also can be occupied either by isoleucine or threonine. A variety of hemoglobins may be formed (reviewed in Kutlar et al., 1989, Hemoglobin 13:671-683 and Honig and Adams, Human Hemoglobin Genetics, Springer Verlag, N.Y. pp. 29-33). Examples include HbA (alpha.sub.2 beta.sub.2), HbA.sub.2 (alpha.sub.2 delta.sub.2), HbF (alpha.sub.2 gamma.sub.2), HbBarts (gamma.sub.4), HbH (beta.sub.4), and Hb PortlandI (zeta.sub.2 gamma.sub.2), Hb Portland II (zeta.sub.2 beta.sub.2), Hb Portland III (zeta.sub.2 delta.sub.2) Hb Gower I (zeta.sub.2 epsilon.sub.2), and Hb Gower II (alpha.sub.2 epsilon.sub.2).
There are obstacles however to using native hemoglobin as a blood substitute. Firstly, large dosages are required (Walder, 1988, Biotech '88, San Francisco, Nov. 14-16, 1988). A single unit (450 ml) of a 10% hemoglobin solution contains 45 g of protein. It is estimated that at least 12 million units of blood are used in the U.S. per year. Therefore the production of 450,000 kg of hemoglobin per year would be required. Secondly, it is important to obtain hemoglobin that is free from infectious agents and toxic substances. Thirdly, as mentioned, although hemoglobin is normally a tetramer of 64,000 molecular weight, it can dissociate to form alphabeta dimers. The dimers are rapidly cleared by the kidneys and the residence time is much too short for cell-free hemoglobin to be useful as a blood substitute. Fourthly, cell-free hemoglobin has too high an oxygen affinity to effectively release oxygen to the tissues due to the absence of 2,3-diphosphoglycerate (2,3-DPG). Efforts to restore 2,3-DPG have been unsuccessful since 2,3-DPG is rapidly eliminated from the circulation.
Several approaches have been taken to circumvent these difficulties. These include the expression of hemoglobin via recombinant DNA systems, chemical modification of hemoglobin, and the production of hemoglobin variants.
2.1.1. EXPRESSION OF RECOMBINANT HEMOGLOBIN
Human embryonic zeta-globin (Cohen-Sohal, 1982, DNA 1:355-363), human embryonic epsilon-globin (Baralle et al., 1980, Cell 21:621-630), human fetal gamma-globin (Slightom et al., 1980, Cell 21:627-630), human adult delta-globin (Spritz et al., 1980, Cell 21:639-645), human adult alpha-globin genomic DNA (Liebhaber et al., 1980, Proc. Natl. Acad. Sci. U.S.A. 77:7054-7058) and human adult beta-globin cDNA (Marotta et al., 1977, J. Biol. Chem. 252: 5040-5053) have been cloned and sequenced.
Both human adult alpha- and beta-globins have been expressed in bacterial systems. Nagai et al. (1985, Proc. Natl. Acad. Sci. U.S.A. 82:7252-7255 and 1984, Nature (London) 309:810-812) expressed adult beta-globin in E. coli as a hybrid protein consisting of the 31 amino-terminal residues of the lambda cII protein, an Ile-Glu-Gly-Arg linker, and the complete human adult beta-globin chain. The hybrid was cleaved at the single arginine with blood coagulation factor Xa, resulting in the liberation of the beta-globin chain. PCT Application No. PCT/US88/01534 (Publication No. WO 88/091799, published Dec. 1, 1988) discloses the expression of a DNA sequence encoding the adult alpha-globin gene and the N-terminal 20 amino acid sequence of beta-globin in which the alpha- and beta-globin sequences are separated by spacer DNA encoding a Factor Xa cleavage site.
Efforts have also been made to secrete beta-globin into the periplasm of E. coli, in which the beta-globin gene was inserted behind an OmpA secretion signal sequence (Brinigar et al., 1988, Symposium on Oxygen Binding Heme Proteins-Structure, Dynamics, Function and Genetics). However, it was found that though the fusion was correctly processed, the beta-globin was not secreted.
Nagai et al. (1985, Proc. Natl. Acad. Sci. U.S.A. 82:7252-7255) have also reported the reconstitution of adult beta-globin expressed in E. coli and adult alpha-globin obtained by conventional sources along with a heme source to obtain hemoglobin. However, it would not be possible in E. coli to produce recombinant hemoglobin that has the same functional properties as normal human hemoglobin because of E. coli's inability to remove the N-formyl-methionine by post-translational processing. The amino terminus is known to be critical in determining the oxygen binding properties of human hemoglobin as has been shown in the case of Hb Raleigh (Moo-Penn, et al., 1977, Biochemistry, 16:4872-4879). Furthermore, the hemoglobin produced in bacteria can contain E. coli endotoxins.
Attempts have also been made to express hemoglobin in yeast. Reports from two groups indicate that yeast cells were unable to excise the intervening sequences in both alpha- and beta-globin precursor mRNA (Langford et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:1496-1500 and Beggs et al., 1980, Nature (London) 283:835-840). An attempt was also made to secrete beta-globin in Streptomyces by constructing a plasmid having a GalK-FX-beta-globin sequence behind a beta-galactosidase secretion signal sequence (Brinigar et al., 1988, Symposium on Oxygen Binding Heme Proteins Structure, Dynamics, Function and Genetics). GalK-FX-beta-globin however remained within the cells under conditions where galactokinase was secreted.
Recently, the construction of two yeast plasmids containing adult beta-globin was reported (Brinigar et al., 1988, Symposium on Oxygen Binding Heme Proteins Structure, Dynamics, Function and Genetics). One contained a constitutive promoter, glyceraldehyde-3-phosphate dehydrogenase and ubiquitin fused directly to adult beta-globin, and the other contained metallothionein, an inducible promoter, and ubiquitin fused directly to beta-globin. It was reported that in both instances, both intracellular soluble and intracellular insoluble adult beta-globin was obtained. No further details were disclosed regarding the construction of the plasmids or the quantity of adult beta-globin obtained.
The expression of globin in mammalian cells has also been reported. The construction of recombinant herpes simplex virus, adenovirus, SV-40, and retrovirus vectors containing a DNA sequence encoding the human adult beta-globin gene has been disclosed (Dobson et al., 1989, J. Virol. 63:3844-3851; Yanagi et al., 1989, Gene 76:19-26; Miller et al., 1988, J. Virol. 62:4337-4345; and Karlsson et al., 1985, EMBO J 5: 2377-2386). The expression of human adult alpha-globin genes in Chinese hamster ovary cells which involved introducing a recombinant DNA molecule containing the normal human adult alpha-globin gene and a hybrid gene containing the 5' promoter-regulator region of the mouse metallothionein gene linked to a SV2-cDNA dihydrofolate reductase gene has also been disclosed (Lau et al., 1984, Mol. Cell Biol. 4:1469-1475). However, the expression of the globin genes was found to be rather low due to low efficiency of gene transfer.
2.1.2. CHEMICAL MODIFICATION OF HEMOGLOBIN
One approach that has been taken to circumvent the problem of dissociation of the hemoglobin tetramer to a dimer has been to chemically modify the hemoglobin by either intramolecular or intermolecular crosslinking. Examples of such modification include crosslinking with polyalkylene glycol (Iwashita, U.S. Pat. Nos. 4,412,989 and 4,301,144), with polyalkylene oxide (Iwasake, U.S. Pat. No. 4,670,417); with a polysaccharide (Nicolau, U.S. Pat. Nos. 4,321,259 and 4,473,563); with inositol phosphate (Wong, U.S. Pat. Nos. 4,710,488 and 4,650,786); with a bifunctional crosslinking agent (Morris et al., U.S. Pat. No. 4,061, 736); with insulin (Ajisaka, U.S. Pat. No. 4,377,512); and with a crosslinking agent so that the hemoglobin composition is intramolecularly crosslinked between lys 99 alpha, and lys 99 alpha.sub.2 (Walder, U.S. Pat. No. 4,598,064).
Hemoglobin has also been chemically modified to decrease the oxygen affinity of isolated hemoglobin. One approach has involved polymerization with pyridoxal phosphate (Sehgal et al., 1984, Surgery, 95:433-438). Another approach has involved the use of reagents that mimic 2,3-DPG (Bucci et al., U.S. Pat. No. 4,584,130). Although these compounds do lower the oxygen affinity of hemoglobin, the affinity is still relatively high.
2.1.3. HEMOGLOBIN VARIANTS
Categories of naturally occuring hemoglobin variants include: variants which autopolymerize, variants which prevent the dissociation of the tetramer, variants with lowered intrinsic oxygen affinity, and variants that are stable in alkali. Examples of autopolymerizing hemoglobin variants include Hb Porto Alegre, Hb Mississippi, and Hb TaLi.
Hb Porto Alegre is a beta chain variant first reported by Tondo et al. (1974, Biochem. Biophys. Acta 342: 15-20; 1963, Am. J. Human Genet. 15:265-279). The beta-9 serine is replaced by cysteine which is able to form disulfide bonds with other cysteine residues. Through these crosslinks, Hb Porto Alegre forms poly-tetramers. These polymers however do not form in the blood of Hb Porto Alegre carriers. It has been shown that Hb Porto Alegre carriers have a two-fold elevated level of glutathione and three-fold elevated level of glutathione reductase which prevents the polymerization of the Hb Porto Alegre within the red blood cells (Tondo et al., 1982, Biochem. Biophys. Res. Commun. 105:1381-1388). The exact structure of these polymers is not known.
Hb Mississippi is a recently isolated polymerizing variant of hemoglobin. The new variant was first reported by Adams et al. (1987, Hemoglobin 11:435-452). The beta-44 serine is replaced by cysteine in this variant resulting in inter-tetramer disulfide bonds. This variant is believed to form polymers with as many as ten tetramers.
Hb TaLi is another known polymerizing beta variant. The beta-83 glycine is replaced by cysteine. This variant was first reported in 1971 (Blackwell et al., 1971, Biochem. Biophys. Acta 243:467-474). This variant also forms inter-tetramer crosslinks.
Another group of variants include those with nondissociating tetramers. One example is Hb Rainier, a well characterized variant of the beta chain (Greer and Perutz, 1971, Nature New Biology 230:261 and Statoyannopoulos et al., 1968, Science 159:741). The beta-145 tyrosine is replaced by cysteine. This cysteine is able to form disulfide crosslinks with beta-93 cysteine which is present in natural beta-globin. This disulfide bond is intra-tetramer, i.e. it is formed between the two beta subunits within a tetramer. This covalent disulfide bond stabilizes the tetramer form and prevents the dissociation of the tetramer into its constituent dimers. Hb Rainier has also been found to have a high affinity for oxygen, a reduced Hill coefficient, and only half the alkaline Bohr effect of normal hemoglobin.
Another group of variants includes those that are stable in alkali. Hb Motown/Hacettepe is a variant reported to be stable in alkali (Gibb and Rucknagel, 1981, Clinical Research 29:795A and Altay et al., 1976, Biochem. Biophys. Acta 434:1-3). The beta-127 glutamine is replaced by glutamic acid in this variant. This portion of the beta chain is involved in the alpha.sub.1 beta.sub.1 interface between the monomers forming a dimer. The substituted glutamic acid forms an ionic bond with alpha-31 arginine. This is a stronger bond than that formed between the alpha-31 arginine and the normal beta-127 glutamine and is believed to be responsible for the increased stability of Hb Motown/Hacettepe. HbF (fetal hemoglobin) and bovine hemoglobin are also in this group of alkali stable variants (Perutz, 1974, Nature 247:341).
There are also over 30 naturally occurring hemoglobin variants which exhibit lowered oxygen affinity. Several examples of such variants are disclosed in PCT Application No. PCT/US88/01534 (Publication No. WO 88/091799, published Dec. 1, 1988); Bonaventura and Bonaventura, 1980, In: Abnormal Human Hemoglobins and Red Cell Enzymes, Huisman, T., Ed., Marcel Dekker, N.Y., Hemoglobin 4 (3 & 4):275-289 and Bonaventura and Bonaventura, 1978, in Biochemical and Clinical Aspects of Hemoglobin Abnormalities, Caughey, W. S., Ed., Academic Press, N.Y., pp. 647-663. There seems in a group of these low oxygen affinity mutants to be a generalizable relationship between the intrinsic oxygen affinity of an alpha.sub.2 beta.sub.2 hemoglobin and the cluster of positively charged residues that are involved in the binding of 2,3-DPG and other anionic allosteric cofactors of hemoglobin function (Bonaventura and Bonaventura, 1980, Amer. Zool. 20:131-138).
One example of a low oxygen affinity mutant is Hb Chico where the beta-66 lysine is replaced by threonine (Shih et al., 1987, Hemoglobin 11: 453-464). The P.sub.50 of Hb Chico's red blood cells is 38 mm Hg compared with normal red blood cell controls with P.sub.50 of 27 mm Hg. All other properties, i.e. Hill coefficient and alkaline Bohr effect are normal.
Another low oxygen affinity variant is Hb Raleigh, a beta chain variant in which beta-1 valine is replaced by alanine (Moo-Penn et al., 1977, Biochemistry 16:4873). A post-translational modification of the amino-terminal alanine results in the formation of acetylalanine. Because the positively charged amino group of valine is involved in 2,3-DPG binding, the acetylation results in a decreased charge cluster in the DPG binding site. This charge difference acts to decrease the oxygen affinity of Hb Raleigh and to lessen the effect of DPG which lowers the oxygen affinity of normal HbA. The Hill coefficient (cooperativity) and alkaline Bohr effect (pH dependent oxygen binding) are unaffected by this change.
Hb Titusville (alpha-94 aspartate to asparagine) is one of a group of low affinity hemoglobin variants with altered alpha.sub.1 beta.sub.2 contacts (Schneider et al., 1975, Biochem. Biophys. Acta 400:365). The alpha.sub.1 beta.sub.2 interface is stabilized by two different sets of hydrogen bonds between the alpha and beta subunits. One set stabilizes the T-structure which is the low-affinity form and the other stabilizes the R-state which is the high affinity form. It is the shifting back and forth between these two sets of bonds and alternating between the T- and R-states which is responsible for the positive cooperativity. The deoxyhemoglobin is primarily in the T-state. For hemoglobin with one oxygen bound, the amount of R-state molecules increases and therefore binds oxygen with a higher affinity. In hemoglobin with two oxygens bound, there is an even higher proportion of R state molecules. In Hb Titusville, the R-state bonds are disrupted. The alpha-94 aspartate would normally form a non-covalent bond with beta-102 asparagine. Because this bond is disrupted, the equilibrium is pushed in the direction of the T-state and Hb Titusville's oxygen affinity is very low.
Hb Beth Israel is another variant affecting the alpha.sub.1 beta.sub.2 interface which destabilizes the high oxygen affinity R-state (Nagel et al., 1976, New Eng. J. Med. 295:125-130). The beta-102 asparagine is replaced by serine. The whole blood of an Hb Beth Israel patient has a P50 of 88 mm Hg as compared with the normal value of 27. The Hill coefficient is biphasic with a value of 1.0 at the high end and 1.8 at the low end. The Bohr effect is normal. A hemolysate of Hb Beth Israel has a P50 of 17 mm Hg and a Hill coefficient of 1.65 at the bottom and 1.29 at the top of the curve as compared to a P50 of 5.6 and a Hill coefficient of 2.72 for normal hemoglobin.
Another example of a low affinity human hemoglobin mutant is Hb Kansas (PCT Application No. PCT/US88/01534, Publication No. WO 88/091799, published Dec. 1, 1988 and Bonaventura and Riggs, 1968, J. Biol. Chem. 243: 980-991). The beta-102 asparagine is replaced by threonine. It has been shown that isolated Hb Kansas' heme-containing beta-globin chains have lowered oxygen affinity (Riggs and Gibson, 1973, Proc. Natl. Acad. Sci. U.S.A. 70:1718-1720).
2.2. EXPRESSION OF HETEROLOGOUS DNA IN YEAST
With the advent of recombinant DNA technology, efforts have been made to express heterologous DNA in a variety of prokaryotic and eukaryotic systems. One such system is yeast.
Yeast has a number of advantages over bacteria and other eukaryotes as a system for the production of polypeptides or proteins encoded by recombinant DNA. Yeast has been used in large scale fermentations for centuries, so the technology for fermenting yeast is well known and a number of yeast hosts are commercially available. Additionally, yeast can be grown to higher densities than bacteria and many other types of eukaryotic cells, and is readily adaptable to continuous fermentation processing. Since yeast is a eukaryotic organism, yeast may be capable of glycosylating expression products, may exhibit the same codon preferences as higher organisms, and may remove the amino terminal methionine during post-translational processing.
A number of heterologous proteins have been expressed in yeast. Examples include interferon (Hitzeman and Leung, U.S. Pat. No. 4,775,622, issued Oct. 4, 1988; Hitzeman et al., Canadian Patent No. 1,205,026, issued May 27, 1986; Hitzeman et al., 1981, Nature (London) 293: 717); platelet derived growth factor (Murray et al., U.S. Pat. No. 4,801,542, issued Jan. 31, 1989); glucagon (Norris et al., U.S. Pat. No. 4,826,763, issued May 2, 1989).
Heterologous proteins expressed in yeast have been linked to a wide variety of promoters. Examples include operably linking heterologous proteins to SV40 and RSV promoters (Gelfand et al., U.S. Pat. No. 4,8710,013, issued Sep. 26, 1989). Additionally, DNA sequences encoding heterologous proteins have been linked to yeast promoters, which are inducible. European Patent Application Publication No. 132,309, published Jan. 30, 1985 discloses the construction of a plasmid containing the yeast galactose-induced promoters for galactokinase (GAL1) and UDP-galactose epimerase (GAL10), hereinafter referred to as the GAL1-10 promoter, which is bidirectional. Another example of a bidirectional yeast promoter is the YPT1/TUB2 intergene sequence which contains overlapping binding sites for the transcription factor BAF1 (Halfter et al., 1989, EMBO J. 8:3029-3037). Broach et al. (Manipulation of Gene Expression, ed. Inouye, 1983) disclose a plasmid containing a GAL10 upstream activator sequence which promotes transcription and an alcohol dehydrogenase transcription (ADH1) terminator sequence to prevent run through transcription derived from YEp51. Kingsman et al., U.S. Pat. No. 4,615,974, issued Oct. 7, 1986 disclose the use of the 5' regions of the yeast phosphoglycerate kinase genes as a promoter of the transcription of interferon. Hitzeman et al., Canadian Patent No. 1,205,026, issued May 27, 1986 disclose the use of the 5' flanking sequence of the ADH1 structural gene to promote the transcription of interferon. Burke et al., U.S. Pat. No. 4,876,197, issued Oct. 24, 1989 disclose a DNA construct comprising a first transcription regulatory region obtained from the yeast lcohol dehydrogenase II gene (ADH2), the regulatory region of acid phosphatase (PHO5) or the regions regulated by GAL4, which provides for inducible transcriptional regulation and a second transcriptional initiation region from the yeast glyceraldehyde-3-phosphate dehydrogenase gene (TDH3) and a terminator region.